Translation of the genetic code from mRNA into protein ultimately determines the protein composition of the cell. In all three kingdoms, translation efficiency and fidelity are modulated by the choice of synonymous codons used to encode polypeptides, although synonymous codons specify insertion of the same amino acid. The problem of how codon choice affects translation is central to biology, but has been difficult to study in part, because the identities of the codons or codon combinations that cause decreased translation have been largely unknown, particularly in eukaryotes. To this end, we performed the first systematic analysis to identify codons that cause decreased translation efficiency in the yeast Saccharomyces cerevisiae, finding that the Arg CGA codon is translated extremely inefficiently, due to I*A wobble decoding interactions in the ribosome, and that CGA-CGA codon pairs are far more inhibitory than isolated CGA codons, implying that other inhibitory pairs composed of non-identical codons exist. To identify such inhibitory pairs, we developed a method coupling fluorescent reporters and deep sequencing, with which we identified 14 strongly inhibitory codon pairs. In examining the mechanism of inhibition by CGA codon pairs, we found that strains lacking the ribosomal protein Asc1 read through CGA codons more efficiently, and also surprisingly undergo extensive frameshifting at CGA repeats. Its human homolog Rack1 has a central regulatory role in signaling pathways. We propose here to follow up with three aims. First, we will determine how the 14 newly identified inhibitory codon pairs impair translation. To define these mechanisms, we will examine the roles of known quality control pathways in inhibition by these pairs, we will identify new genes involved in codon-mediated inhibition by selecting mutants with improved decoding of inhibitory codon pairs, and we will ascertain whether or not ribosomes stall at these codon pairs. Second, we propose to investigate the role of Asc1 and other proteins in reading frame maintenance, by identifying mutations in other genes that independently cause frameshifting at CGA repeats, or that participate with Asc1 in preventing frameshifting. Third, to assess the functional role(s) of inhibitory codon pairs, we will determine if conservation of these codon pairs is related to their inhibitory function by comparing conservation of these inhibitory codon pairs in species with similar and different decoding strategies from S. cerevisiae. We will also evaluate the consequences of altering inhibitory codon pair function, both in the entire organism by examining the effects of expressing non-native suppressor tRNAs on yeast fitness, and in specific genes by investigating effects of mutating their specific inhibitory codon pairs on their expression, function, RNA structure and variability of expression. Completion of these aims is highly likely to result in significant insights into the fundamental question of how ribosomes regulate gene expression, and has high promise for uncovering global regulatory mechanisms that are conserved in evolution.